Apparatus for structural characterization of biological moieties through HPLC separation

ABSTRACT

A system allows routine coupling of HPLC separations to sheathless μ-ESI sources for MS analysis. Characteristics of this system include stable electrospray throughout the HPLC gradient at low and high flow rates, lower background than conventional sources, and control over the width of eluting HPLC peaks without degradation of HPLC performance. This system includes a pre-column solvent flow splitter, a metal union in the split waste line for application of μ-ESI voltage, a divert valve containing two different size restrictors for control of flow, and pulled fused silica capillaries as μ-ESI emitters. The pulled tips allowed stable operation of the system with column flow rates ranging from ca. 5-&gt;250 nL/min.

This application is a regular National application claiming priorityfrom Provisional Application, U.S. application Ser. No. 60/062,486 filedOct. 20, 1997. The entire disclosure of the provisional application isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to an apparatus and method for characterizingpeptides and other biological moieties by electrospray ionization massspectrometry of samples provided by high performance liquidchromatography (HPLC). A device is provided which permits broadening ofHPLC elution peaks without loss of resolution, simultaneously slowingthe solvent flow of the system without burdening the system withsubstantial dead volume for the HPLC eluent. The device also providesfor application of the voltage necessary for sheathless electrosprayionization in a waste line thereby overcoming issues related toapplication of the voltage.

2. Background of the Prior Art

Mass spectrometry has rapidly developed as the method of choice forsequencing biologically derived molecules.¹,2 The high complexity ofbiological mixtures often makes coupling a separation technique, such asHPLC, highly desirable or even required.² Unfortunately, for manydetection techniques including mass spectrometry, as the separationefficiencies increase, the peak widths tend to narrow placing morestringent speed requirements on the mass spectrometer (MS), makingstructural analysis of peptides by collisionally-activated dissociation(CAD) difficult.

Also important in many biological analysis is the detection limit ofelectrospray ionization MS techniques. Electrospray ionization (ESI)efficiencies with conventional ESI sources are compromised in two ways.First, as an analyte enters into the ionization region of the ESI sourceit is diluted by a sheath liquid to help stabilize the ESI process andapply the potential needed for ESI to the HPLC effluent.³ Second, thelarge electrospray plume generated by the convention ESI source issampled by a very small orifice leading into the MS giving lowtransmission efficiencies.⁴ Recent advances in ESI techniques have shownimprovements in ESI transmission efficiencies by eliminating sheathliquids and reducing total flows into the ESI source.³⁻⁸

One of the most widely used miniaturized sheathless ESI sources (μ-ESI)is the Nanospray source of Mann et al.⁵ This source uses glasscapillaries pulled to <5 μm, sample flow rates at <5-50 nL/min, and iscapable of analyzing low femtomole amounts of sample in a volume of 1 μLcontinuously for more than 1 hour. The long analysis time allowsmultiple MS experiments to be performed. This method, however, oftenrequires substantial signal averaging (>10 min) to acquire satisfactoryS/N to identify precursor masses for subsequent CAD experiments and doesnot have the ability to interface with a separation technique. The tipsfor this source are fragile, expensive, non-reusable, and the positionof the tip at the orifice into the mass spectrometer is critical, oftenrequiring expensive camera equipment in order to achieve optimum signal.

The use of reverse phase HPLC on line with μ-ESI for the analysis ofpeptide mixtures offers numerous advantages over Nanospray ESI,including desalting and detergent removal, complicated mixtures can betime resolved, more dilute samples can be used because the sample isconcentrated on column, and information on the hydrophobicity of theanalyte can be obtained. However the short time window in which apeptide elutes can be problematic if multiple stages of MS are desired.In order to sequence biological peptides using MS, it is necessary to 1)identify the peptide and 2) dissociate the peptide such that itfragments randomly along the amide linkages. CAD on the ion trap MSinvolves several steps. The precursor ion must first be isolated usingtailored rf waveforms applied to the endcap electrodes. The ion mustthen be activated by increasing the ions kinetic energy using rfwaveforms applied to the endcaps in resonance with the ion in thepresence of a bath gas such that the ion collides with the bath gasconverting kinetic energy into internal energy. The ion then mayfragment and the fragments are scanned out and detected. Thisidentification/CAD process can be 5 seconds while typical HPLC peakwidths are ca. 12 seconds.

It has been shown that slowing the flow rate down during an HPLC run cansatisfactorily increase the elution time of a peptide by as much as 10times.⁷,8 Potential problems with this method for commercial syringepump HPLC systems are the time delay before the slow column flow isrealized and the dead volume of the system after slow column flow isachieved. The time delay before slow column flow is achieved arises fromthe total volume of the system being at the running pressure of thecolumn. To slow down the column flow the entire HPLC system pressuremust be reduced to the desired low column flow pressure. The secondpotential problem is the dead volume after the solvent mixing tee. Thedesired HPLC gradient must pass through the dead volume after the mixingtee at the slow flow rates, this will give significant lag times in theHPLC gradient profiles. Davis et al. use a preformed gradient in a fusedsilica capillary (FSC) and a programmable ISCO syringe pump to slow downand speed up the flow rate of the mobile phase over the column.⁷ Tocombat the lag time before slow flow is achieved they actually reversethe syringe pump to lower the column head pressure very quickly. Thepreformed gradient fixes the profile of the HPLC run before changingpressures such that they have no HPLC gradient lag times due to deadvolumes. Unfortunately, these methods to overcome dead volume issueswill not work with more common HPLC syringe pump systems.

Accordingly it remains an object of those of skill in the art to developa process to characterize biological molecules and compounds by MS,particularly, through ESI MS. It is a further object of those of skillin the art to provide an apparatus that permits broadening of HPLC peaksin an ESI environment, without dead volume and at a slow or controlledflow rate.

SUMMARY OF THE INVENTION

The above objects, and others described in more detail below, are met byproviding an apparatus comprising an HPLC column joined with an ESIemitter, which in turn transmits the eluting sample to a MS or otherdetector. The line for introducing the mobile phase to the column issplit at the head of the column, one portion continuing on to passthrough the column to perform the analytical separation, and the secondportion constituting a "waste stream" across which the voltage for ESIemission is applied, and which, by controlling the diameter of the exitfrom the waste stream, permits control over the flow speed of the mobilephase. By slowing down the mobile phase, the elution peak for anyspecific moiety may be broadened, permitting thorough characterizationof the same by the detector.

Described below is a method for performing a similar experiment usingtwo different lengths of fused silica capillary as restrictors whichcontrol the column flow between fast (100->200 nL/min) and slow (<10-30nL/min) mobile phase flow rates using a common commercial HPLC syringepump system. The mobile phase can be switched to low flow as a peptidebegins to elute, broadening elution peaks sufficiently to allow MS^(n)experiments to be performed on multiple analytes in a single HPLC run,even when the masses of the peptides are not known ahead of time.

In order to couple HPLC with a sheathless μ-ESI source, a method must beprovided to supply the μ-ESI potential to the HPLC effluent. Currently,there are two methods for applying the potential needed for μ-ESI to theμ-ESI emitter tip: direct contact though metal emitters or metalizedtips and liquid junctions.³,6-9 Metal emitters suffer from backgroundattributable to the metal, while metalized tips have limited lifetimesof often less than one day. Liquid junctions typically have issues withbackground ions due to the metal of the liquid junction or instabilitydue to formation of bubbles in or past the junction. Here we present anovel liquid junction for coupling HPLC with μ-ESI overcoming peak widthlimitations, chemical noise problems, and stability issues.

The apparatus includes a standard HPLC column which is provided with asplit or "T" at the head of the column, between the column itself andthe eluant reservoir/pump. The waste column is equipped with a metalcontact, or applied charge across the column, for providing thepotential for the ESI emitter at the end of the HPLC column. A MS isadjacent to the ESI capillary. By controlling the flow rate of the wastecolumn, preferably with a multi-opening diverter, the flow rate can becontrolled and HPLC peaks broadened to provide opportunities forrepeated sampling, more MS experiments and therefor completecharacterization of biological molecules of interest being eluted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an HPLC-μESI system used for controlling the elutiontimes of peptides from reverse phase column. With the valve in position1-2, the mobile phase flow rate over the column is 100-200 nL/min.,giving HPLC peak widths of 10-25 seconds. When the value is in position1-3, the flow rate over the column is typically 10-30 nL/min., producingHPLC peak widths of 1.5-5 min.

FIG. 2A illustrates a base peak ion chromatogram.

FIG. 2B illustrates an ion chromatogram for 100 fmol of Brodykinin.

FIG. 2C illustrates an ion chromatogram for 100 fmol of Substance P.

FIG. 2D illustrates an ion chromatogram for 100 fmol of Des-R⁹-Bradykinin.

FIG. 2E illustrates an ion chromatogram for 100 fmol of FibrinopeptideB.

FIG. 2F illustrates an ion chromatogram for 100 fmol of Dynorphine A(1-13).

FIG. 3A illustates a base peak ion chromatograms.

FIG. 3B illustrates an ion chromatogram for 100 fmol of Brodykinin.

FIG. 3C illustrates an ion chromatogram for 100 fmol of Substance P.

FIG. 3D illustrates an ion chromatogram for 100 fmol of Des-R⁹-Bradykinin.

FIG. 3E illustrates an ion chromatogram for 100 fmol of FibrinopeptideB.

FIG. 3F illustrates an ion chromatogram for 100 fmol of Dynorphine A(1-13).

In each of the above Figures, the HPLC system was switched from highflow (100-200 nL/min.) to low flow (10-30 nL/min.) at 3.3 min. (arrow Ssand then switched back to high flow at 6.1 min. (arrow f).

These chromatograms clearly show th eability of the system to rapidlyslow down the flow over the column resulting in broadening of elutiontime.

FIGS. 4A-4F illustrates, respectively, ion chromatograms of the samepeptides as in FIG. 2.

These Figures demonstrate the ability to slow the flow and, thus,increase the elution times of multiple peaks. The arrows indicate timeswhen the value position was switched from high flow (arrow s) or lowflow to high flow (arrow f).

FIGS. 5A-5H illustrate the high sensitivity of the HPLC-μFSI system.Horse heart Cytochrome C was digested with trypsin and the equivalent of1.5 fmol of Cytochrome C was analyzed.

The selected ion chromatograms are shown for base peak and eight (8)tryptic fragments of Cytochrome C, with the corresponding residuenumbers of each peptide being indicated.

This experiment was performed with the system in high flow (100-200nL/min.) mode throughout the entire gradient. The peak widths of thepeptides are typically 20 seconds.

FIGS. 6A-6B illustrates CAD spectra for two tryptic fragments ofCytochrome C as indicated.

The equivalent of 1.5 fmol of Cytochrome C was analyzed using theHPLC-μESI system described. The system was run at high flow mode with apeptide of interest being observed in the MS spectrum. The flow was thenrapidly switched to slow flow and CAD spectra were collected. Uponcomplete acquisition of CAD information, the flow was returned to highflow until the next peptide eluted.

Five CAD spectra were collected with ease without pre-programming orpre-selecting the peptides to be analyzed by CAD. All five CAD spectraindicated Cytochrome C as the parent protein when searched using MS-Tap.

This system has been used to collect thousands of CAD spectra in one runwithout pre-selection of the peptides to be analyzed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention embraces an apparatus for controlling the flow rate ofHPLC elution, and broadening the peaks thereof, so as to permitcharacterization of molecules, particularly biological ones, in theelution fraction, through ESI MS. By carefully controlling eluant flow,while controlling for dead volume, precise characterization of themolecules eluted may be obtained.

The apparatus is illustrated in FIG. 1. The mobile phase enters theapparatus 100 at a point upstream 102 from the actual HPLC column,coming from the mobile phase pumps (not pictured). This entry point isconventional polyetheretherketone (PEEK) tubing 104 or other suitablefluid conduit. This tubing is fitted into a fitting (PEEK ferule) 106which holds the tubing in tight association with the HPLC column 110through a "T" fitting 108 which splits the flow into two streams, oneentering the column 110 and one referred to as the "waste" stream 112,which permits control over flow rate. The column itself comprises tubing110 joined through a connector 116 to an ESI emitter 130 which may be anFSC or may be a metallic tip if the charge potential is not applieddirectly to the tip. In a preferred embodiment, voltage for the electricpotential is applied to metal fittings or union 118 across the wasteline or stream 112.

In a preferred embodiment, the apparatus of the invention is providedwith a diverter 120, as shown in FIG. 1. In this embodiment, thediverter valve may occupy multiple positions corresponding to openingsof different diameter 122 and 124 which give different flow rates,permitting differential broadening of HPLC peaks at different stages ofthe elution. In this embodiment, the diverter opening may comprise PEEKtubing 126 coupled with capillaries 128 of different volume.

The apparatus is used to perform HPLC-ESI mass spectrometry of peptidesand other biological compounds, based on elution pattern. The mobilephase enters the waste stream and column head, the waste stream slowingcolumn flow and controlling for "dead volume" in advance of actualpassage through the HPLC column. By controlling the speed of flow in thewaste stream, by varying the diameter of the exit opening therefore, thespeed of passage of the mobile phase may be retarded, and therefore the"elution peak" broadened, to permit repeated sampling, fragmentation andcharacterization of the biological moiety in question. The invention maybe further understood by reference to the Examples set forth below,which are not intended to be limiting.

Experimental

HPLC columns were prepared by packing 5 cm of a 7 cm×360 m.×75 m. fusedsilica capillary (F.C., Polymicro Technologies, Phoenix, Ariz.) with 10m. C-18 beads YMC (Wilmington, N.C.) as reported previously.⁹ HPLCgradients were formed using an ABI (foster city, Calif.) 140B syringepump system. Gradients were 0-60% B (A=0.1 M CH₃ COOH in H₂ O, B=CH₃ CN)in 19 min. The 200 μL/min flow from the syringe pump was split at thehead of the column using a Swagelock ZDV tee (Richmond, Va.) with thewaste passing through a titanium ZDV union (Valco, Houston, Tex.) theninto a Valco Six Port two position divert valve (Finnigan, San Jose,Calif.) as shown in FIG. 1. The divert valve regulated the column flowby allowing switching between two different FSC. restrictors located inthe mobile phase waste line. The restrictors were chosen to give thedesired column flow and peak broadening, and were typically 360 m.×50m.×10 cm and 280 m.×100 m.×5 cm for the high and low flow ratesrespectively. Column flow rates were generally switchable betweenca. >150 nL/min and <20 nL/min.

ESI emitters were prepared by hand pulling 360 m.×50 m. FSC using themethod of Davis and Lee.⁷ Briefly, a 400 g weight was suspended from theFSC. The FSC was then heated with a bright blue flame produced by amodified Microflame micronox torch (Mennetonka, Minn.). The torch wasmodified by replacing the flame tip with a 22 gauge stainless steelneedle. The pulled FSC filaments were then cut to the desired o.d./i.d.under a microscope using a razor blade and butt connected to the end ofthe HPLC column using a piece of Teflon drilled to 368 m. i.d. as shownin FIG. 1.

ESI voltage was applied to the metal union in the split waste lineutilizing the conductivity of the HPLC mobile phase to get the potentialto the emitter tip. Typical voltages were 1.4-2 kV dc. The distance ofthe metal union to the tip of the μ-ESI emitter was ca. 18 cm. Thedistance of the emitter to the heated capillary orifice was ca. 1 mm.

All analysis were performed on a LCQ (Finnigan MAT, San Jose, Calif.)ion trap mass spectrometer. Spectra were collected with automatic gaincontrol (AGC) on, AGC targets of 2×10⁷ and 7×10⁷ for MS and Ms^(n) scanmodes respectively, and maximum injection times of 500 ms. AGC is usedto control the number of ions that are stored in the trap to overcomespace charge limitations. A scan on the LCQ consists of both an AGCprescan and an analytical microscan, each repeated n times (3 in ourcase) as specified by the user. The AGC prescan determines the ion fluxin 3 ms and then calculates the injection period required to give theAGC target values requested by the user. All spectra were recorded incentroid mode using a mass range of 300-2000 m/z. Database searches wereperformed using the MS-Tag program (http://rafael.ucsf.edu/) with theSwiss-Prot database.

The peptide standards (bradykinin, substance P, des-R⁹ -bradykinin,fibrinopeptide B, and dynorphin A(1-13), Sigma Chemical Co., St. Louis,Mo.) were made by adding the appropriate amount of peptide to 1% CH₃COOH, 5% CH₃ CN to give a final concentration of ca. 1 nmol/μL in 1 mL.Each of these peptide standards were then diluted in 1 mL 1% CH₃ COOH togive a standard peptide mixture containing five peptides at aconcentration of ca. 1 pmol/μAL each. 250 pmol of Horse Heart CytochromeC from Sigma (St. Louis, Mo.), was digested overnight with Promega(Madison, Wis.) modified trypsin, in 40 μl of ammonium acetate (pH=8.0)at a protein:enzyme ratio of 25:1. The Cytochrome C samples describedbelow were dilutions of this digest.

Results and Discussion

FIGS. 2-4 show selected ion current chromatograms recorded for theseparation of 5 standard peptides. FIG. 2 shows the separation and peakwidths obtained without switching to low flow rate. FIG. 3 demonstratesthe resultant peak broadening after reducing the flow and the subsequentnarrowing of the peaks with a return to high flow rate. Peak widthsbefore broadening were ca. 24 seconds. After reducing the flow, the peakwidth of the analyte peak was increased to ca. 3 minutes. The width ofthe peak after reducing the flow is dependant on the ratio of the flowbefore reduction to that of the flow after reduction. The stability ofthe low flow μ-ESI is the only factor limiting the reduction in flow andtherefor the peak width. For these experiments the pulled F.C. were usedto increase the stability of low flow μ-ESI operation. Stable μ-ESI waseasily achieved at flow rates of between 10 and 20 nL/min afterreduction while maintaining stable high flow operation. FIG. 4illustrates the ability to resume a HPLC run after a slow flowexperiment and slow the flow for another later eluting analyte.

FIG. 5 shows the selected ion chromatograms for several peaks seen in aHPLC separation of a tryptic digest of Cytochrome C at the 1.5 fmollevel. This illustrates the ability of the system to afford excellentseparations at low sample levels. FIG. 6 shows two examples of the highquality MS/MS CAD spectra obtained using this system at these low samplelevels. Five of the CAD mass spectra were input into the MS-Tag proteindatabase search program. Each of these spectra gave Cytochrome C as theparent protein.

We have shown a system capable of rapid switching between HPLC flowrates. This system was inexpensive, easy to construct, required nomodifications to current HPLC systems, and is fully controllable. Thesystem provided stable electrospray, provided less background ions thanother methods of μ-ESI voltage application and did not adversely affectHPLC separations. Routine application of sheathless μ-ESI coupled to MSdetection will benefit from this setup.

This method of increasing peak widths can easily be applied to anydetection technique (NMR, ESR, etc.) benefitting from longer analysistimes for an eluting HPLC peak.

This invention has been described in generic terms, and by reference tospecific embodiment. Specific embodiments, examples and experiments arenot intended to be limiting, and variations will occur to those of skillin the art without the exercise of inventive faculty. Variations incomposition, detector means, specific equipment and the like remainwithin the scope of the invention, unless excluded by the claims setforth below.

REFERENCES

1. Biemann, K. Annu. Rev. Biochem. 1992, 61, 977-1010.

2. Cox, A. L.; Skipper, J.; Chen, Y.; Henderson, R. A.; Darrow, T. L.;Shabanowitz, J.; Englehard, V. H.; Hunt, D. F.; Slingluff, C. L. Jr.Science 1994, 264, 716-719.

3. Wahl, J. H.; Gale, D. C.; Smith, R. D. J. Chromatogr. A 1994, 659,217-222.

4. Wilm, M. S.; Mann, M. J. Mass Spectrom. Ion Processes 1994, 136,167-180.

5. Wilm, M. S.; Mann, M. Anal. Chem. 1996, 68, 1-8.

6. Kriger, M. S.; Cook, K. D.; Ramsey, R. S. Anal. Chem. 1995, 67,385-389.

7. Davis, M. T.; Stahl, D. C.; Lee, T. D. J. Am. Soc. Mass Spectrom.1995, 6, 571-577.

8. Davis, M. T.; Stahl, D. C.; Hefta, S. A.; Lee, T. D. Anal. Chem.1995, 67, 4549-4556.

9. Hunt, D. F.; Alexander, J. E.; McCormack, A. L., Martino, P. A.;Michel, H.; Shabanowitz, J.; Sherman, N.; Moseley, M. A.; Jorgenson, J.W.; Tomer, K. B. Techniques in Protein Chemistry II; J. J. Villafranca,Ed.; Academic Press, New York, 1991; pp. 441-454.

What is claimed is:
 1. An apparatus for effecting electrosprayionization (ESI) of liquid material eluted from a high performanceliquid chromatography (HPLC) column while providing for control overspeed of flow of mobile phase through said column, comprising:a conduitfor elution mobile phase which is in fluid communication with a junctionwhich splits said mobile phase into a first steam to enter said columnand a second waste stream, an HPLC column in fluid communication withsaid junction for receiving said mobile phase and eluting from saidcolumn a moiety of interest, an ESI emitter at an end of said columndownstream from said junction and in fluid communication therewith,electrodes for applying an electrical current across said waste stream,a device configured to control the speed of flow of said mobile phasethrough said column, whereby an elution peak over which said moiety iseluted may be broadened, as opposed to a peak obtained in the absence ofsaid waste stream.
 2. The apparatus of claim 1, wherein said apparatusis for obtaining a characterization of a biological moiety, and said ESIis in communication with a detector for detecting characteristics ofsaid biological moiety.
 3. The apparatus of claim 2, wherein saiddetector is a mass spectrometer, a nuclear magnetic resonance detector,or an electron spin resonance detector.
 4. The apparatus of claim 1,wherein said device configured to control the speed of flow of saidmobile phase through said column comprises an opening in said wastesteam downstream of said junction which may be varied between differentdiameters so as to permit control over said speed of flow.
 5. Theapparatus of claim 1, wherein said ESI comprises a fused silicacapillary (FSC) having an outside diameter, at its downstream tip, offrom 0.5-10 microns.
 6. The apparatus of claim 5, wherein saidelectrodes comprise a conductive joint defining, in part, said wastestream.